Note: Descriptions are shown in the official language in which they were submitted.
~ 21~5~
~ W O 94/04541 PC~r/US93/07054
-1-
PROTEIN KINASE INHIBITORS AND RELATED
COMPOUNDS COMBINED WlTH TAXOL
FIELD OF THE INVENTION
This invention describes the use of compounds that are used in combin~tion with taxol
5 to control cancerous growths and tumors. Protein kinase inhibitors and related compounds are
combined with taxol and taxol related coL"~oullds and the combination of compounds exhibits
powerful pot~nti~tin~ effects when used to treat cancer. Many of the coLupo~u,ds are protein
kinase inhibitors, other c(~ .o~ s achieve similar effects but are not nPcess- ily protein kinase
inhibitors.
BACKGROUND OF THE INVENTION
Taxol was first isolated from the bark of the westem yew, Taxus brevifolia, and
ithPntifiPd as an ,.~ ..or agent in 1971 by Wani, et al. Recently, phase II clinical trials with
taxol have shown it to be one of the most exciting chemolllel~euLics available. Taxol has
p;oven effective in drug-,~r,~v,,~ ovarian cancer (McGuire, et al., 1989), and has shown a 56%
15 objective response rate in met~ct~tic breast cancer (Holmes, et al., 1991). In addition, there is
reason for hope that taxol may be effective in many other types of cance~..
The development of taxol, however, has faced many obstacles. Taxol's poor solubility
required that it be a~lminictpred in the vehicle Cremophor EL (polyet'nylated castor oil), which
led to a high inridPnre of hypPrsPncitivity re~chonc. It is not clear whether these reactions are
20 caused by the vehicle or the drug, but it was found that using longer drug i~u~.iO~lS (Weiss, et
al., 1990) and anti-allergic r~gimenc (Rowinsky, et al., 1990) reduced the inridPnre of such
reactions. In addition, there are inherent problems in producing sufficient quantities of taxol.
Extraction from the bark of the extremely slow growing westem yew using present methods
cannot meet the demand for taxol. Cultivation of the westem yew may take years to establish,
25 synthesis of the complex taxol molecule will be difficult andbr very expensive. Altemative
sources of taxol or a taxol ~.I.'~Dlul~ or a taxol additive would therefore be highly desirable.
Taxol has been shown previously to be toxic to tumor and leukemia cells inoculated in
mice, inrluriin~ L-1210, P388 and P-1534 le~lk~mi?~ ceDs and Walker WM-256 carcinosarcoma,
sarcoma 180 and Lewis lung tumor cells (Wani et al., 1971). It has also been shown to be toxic
30 to cultured human HeLa cells (Schiff et al., 1979 ) and CHO (Chinese hamster ovary) cells
(Cabral et al., 1981). This evidence of toxicity to rodent and human tumor cells in vitro and to
tumor bearing mice in vivo predicted that taxol would be an active chemotherapeutic agent and
led to clinical trials in human cancer patients. These clinical trials showed efficacy of taxol in
treating ovarian cancer (McGuire et al., 1989). Taxotere is a taxol type compound that has also
35 been shown to have po~elrul antitumor activity. Bissery et. al, Cancer Research 51, 4845-4852,
Sept. 15, 1991.
2140~ ~
- WO 94/04541 ; ~ PCI/US93/07054
-- -2-
Since taxol is now known to be an effective chemotherapeutic agent, a co-treatment that
incre~ses the toxicity of taxol on cancer or transformed cells, such as CHO cells, would be
likely to increase the chemotherapeutic effect of taxol in cancer patients or to allow smaller
doses of taxol to be ~minicf~red. Qn~ntitjçs of taxol available are extremely limited.
Compounds that increase the efficacy of taxol, thereby allowing smaller amounts to be used
with equal effectiveness, will enable more patients to be treated with taxol. This should also
reduce the hy~ e.~ilivity and non-thc.a~u~c toxic reactions seen clinically with taxol, as both
less taxol and the less of the vehicle needed to deliver taxol will need to be a~mini~tPred.
Our finding is that when the coLupoullds of this invention are com~inP~d with taxol or
taxol related compounds the mixture of coLu;~oullds has a po~nti~tin~ effect that, surprisingly
produces tumor cell toxicity at lower doses than taxol alone. These finrling~, and other studies,
suggest that the COLLII)OUIIdS will be effective in ~yllel~L~LIlg with taxol in killing tumor cells in
human cancer p~tiçnt~ The findings also suggest results may be seen with taxol related
colllpou-lds such as taxotere and related taxol analogues.
INFORMATION DISCLOSURE
Many of the cc,LLl~ullds of this inverltion are related to the physiologically active
subst~n~e K-252. The following patents disclose some of these colllp)ullds. U.S. 4,877,776
issued October 31, 1989. U.S. 4,923,986 issued May 8, 1990. W.O 8807~45-A p~lblichpd
September æ, 1988.
The following Japanese patent applications also disclose related coL~ uullds: J63 295-
588-A, J63 295-589-A, J62 155-28~A, and l62 155-285-A disclose ~ ,olI le relatedcompounds.
SUMMARY OF THE INVENTION
This invention is in two parts. Known co,~ vul,ds are listed in part I, they are claimed
for the method of using the c.JIll~ullds as described herein. The known colllpounds are also
claimed as col,lp~,u,lds combinPd in a c~mposi~ion with taxol type co..l~oullds. The new
-- compounds are in part II. The new colllpvu.l~ls are claimed as col.l~u.lds, for their method of
use and in a composition.
I. The known colll~oullds.
A. Indolocarbazole Type Cou.l)oullds
1) The "First Known Derivatives of K-252." The "First Known Derivatives
of K-252" are all of the compounds (licclosP-d in U.S. patent 4, 877,776. U.S. patent 4, 877,776
incorporated herein by reference.
2) The "Second Known Derivatives of K-252." The "Second Known
3~ Derivatives of K-252" are all of the colll~ullds dicclosed in U.S. patent 4,923,986. U.S. patent
4,923,986 i~-col~ldled herein by reference.
~l ~
21~0~S3
WO 94/04541 PCI /US93/07054
-3-
3) The specific cu,llpoullds below are more preferred,
3(a) KT5823
CH3
~ CH3
~~0 ~CH3
3(b) K-252A
O~_N/
~
H0 , H3C
O=CH38
3(c) KT5926
H3C~ o~_N/
'~ f~'
O=C
C~
- 21~0~
WO 94/04541 PCI /US93/07054
3(d) KT5720
H0 , H3C
O--C
C~2
(C~2)3
CH2
CH2
3(e) Staurosporine
B. Non--n-lolec~rba~ole Type G~ youllds
1) Adriamycin
2) Amiloride
3) Calphostin
4) Chlorpromazine
S) The colllyoulld known as "HA-1004"
6) Indomethacin
7) Okadaic acid
8) PhPn~7ocinP.
9) Polymyxin B
10) 2-~Ulil~OyUlill~
I l) 6-dimethyl-ami-~oyulille
12) Sphingosine
l 3) Tamoxifen
14) Compounds related to tamoxifen such as triphenylethylene antiestrogens
15) Trifluope,d~i-le
16) Verapamil
17) 3-isobutyl-1-methyl-Y~nthin~.
18) 8-Cl-cAMP
~ W O 94/04541 214 0 6 ~ 3 PC~r/US93/07054
II. The new co",poul,ds.
A compound of FORMULA I, below,
~1
~R3
R8 ~R4 FORMULA I
R7 ~,-~`C~
R6 (~
wherein,
Rl is -H, -(Cl-C4 alkyl), -C(O)-(Cl-C4 alkyl), -NH2,
-C(O)-NH2, -CH2CH2-N(Rl-1)2'
wherein Rl l is -H or -(Cl-C4) alkyl,
R2 is -H, or R2 and R3 taken together are (O),
R3 is -H, -OH or R2 and R3 taken together are (O),
R4 is -H, -OH, -NH2, or-0-(Cl-C4 alkyl),
R5 is -OH, -O-(C1-C4 alkyl), or-O-C(O)-(C1-C4 alkyl),
R6 is (C6-C12 alkyl), -(C3-C10 cycloalkyl), -(CH2)nCH2N(R6 1)2
wherein R6 1 is -H, or -(Cl-C4 alkyl),
R7 is -H, or-NH2.
R8 is -Cl, -Br, -H, -CH3, -CH2OH, -OH; -O-(C1-C4 alkyl),
-N(R8 1)2. r-NHC(O)-NH(R8-l)~
wherein R8 1 is -H or-(C1-C4 alkyl)
wherein n is 0-5
with the proviso that:
a) when R2 or R3 is -OH then Rl is H,
b) when R1, R2, R3, R4, and R7 all equal H and R5 is OH
then R6 does NOT equal -(CH2)5CH3,
c) when R1, R4, and R7 all equal H, and R2 combined with R3 is (O),
and R5 is OH, then R6 does NOT equal -(CH2)5CH3.
d) when R4 is -OH, -NH2, or-O(C1-C4 alkyl), then R4 and R8 are the same.
A pharm~ceutic~l composition co~ ~-g of a ph~rm.~reutic:ll1y acceptable carrier and an
effective amount of FORMVLA I. A rh~nn~cel)tic~l composition con.~ in~ of a
2140~
= W O 94/04541 PC~r/US93/07054
-6-
pharm~c~utic~lly acceptable carrier and an effective amount of the col.lpoulld of FORhlULA I
in conjunction with an ~prupliate dose of taxol or taxol related colllpoullds. A method of
controlling cancerous growths in m:~mms~lc which comprises ;lrlminict~oring a ll~l~ulic or
prophylactic dosage of any of the three following groups of coulpoullds in conj~m~tion with an
5 appropriate dose of taxol or taxol related compounds. 1) a compound of FORMULA I, 2) any
one of the co~poul,ds described in the specification as "Indolec~ul,d~ole Type Compounds.", 3)
any one of the COIll~ull~S described in the specification as "Non-lndolecarbazole Type
Compounds."
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Isobologram showing pot~nti~ting effect of the c~nmbin~tion of Taxol
plus KT5823. The isobologram shows the effectiveness of a combination of 2 drugs for the
killing of wild type, 10001a CHO, cells. The data line is the solid line with open circle or
triangle data points. The data line shows the combin~ion of doses which gives an LD50 for the
cells. The diagonal dashed line shows the predicted concelll alions of drugs if meir combination
15 only had an additive effect. If any data points were above the dashed line that would indicate
the combination of colllpoullds had antagonistic effects. Data points below the line indicate the
cn.,.l o...l-is have potenti~ting or synergistic effects.
Figure 2. Isobologram showing potenti~ting effect of the csmbin~tion of taxol plus
KT5926.
Figure 3. Isobologram showing pot~nti~ting effect of the comhin~tion of taxol plus
KT5720.
Figure 4. Isobologram showing NO potentiating effect from the combin~tion of
t3xol plus H-9. This isobologram shows the predicted effect of a "control" subst~nce that does
NOT act in a pot~nti~ting or ~yller~ ic manner.
Figure 5. Isobologram showing pott~nti~ting effect of the combin~fion of taxol plus
K252a
Figure 6. Isobologra_ showing potenti:~ting effect of the combin ~tion of taxol plus
tamoxifen.
Figure 7. Isobologram showing potentiating effect of the combin~tion of taxol plus
30 2-all~h~opu~ e.
Figure 8. Isobologram showing pot~-nti~ting effect of the colllbin~ion of taxol plus
6-dimethylaminopurine.
Figure 9. Isobologram showing pot~nti~ting effect of the cornbin~tion of taxol plus
chlorprnm~7in~
Figure 10. Isobologram showing potenti~ting effect of the comhin~tion of taxol plus
3-isobutyl- I-methyl-x~nthine
-- 21~6S~:
WO 94/04541 PCI /US93/07054
-7-
Figure 11. Isobologram showing potPnti~ting effect of the combin~tion of taxol plus
8-CI-cAMP.
Figure 12. Isobologram showing potenti:~ting effect of the combination of taxol plus
F.~mr~le A-l.
Figure 13. Isobologram showing potpnti~tin~ effect of the combination of taxol plus
Example B-l.
Figure 14. Isobologram ~howu~g potP-nti~tin~ effect of the combination of taxol plus
Example B-2.
Figure 15. Isobologram showing potPnti~ting effect of the c~ ion of taxol plus
10 ~xample B-3.
Figure 16. Isobologram showing potPnti~ting effect of the combin~tion of taxol plus
Example B4.
Figure 17. Isobologram showing potpnti~ting effect of the combination of taxol plus
Example B-5.
Figure 18. Isobologram showing potenti~ting effect of the combination of taxol plus
Example B-6.
Figure 19. Isobologram showing potenh~ting effect of the combination of taxol plus
Example B-7.
Figure 20. Isobologram showing potPnh~ting effect of the combination of taxol plus
20 Example B-8.
~igure 21. Isobologram showing potenti~tin~ effect of the combin~tion of taxol plus
Example B-9.
Figure 22. ~ffect of KT5î20 and taxol on the growth of MX-1 tumors.
Figure 23. Table of data showing toxicity of several of the drugs both individually
25 and in combination with taxol on non-tumored mice. (In Vivo Effects)
DETAILED DESCRIPTION OF THE INVENTION
The c~ll.pou,.ds of this invention are of two types. The first type are known compounds
described here for their usefulness when combinPd with taxol type compou--d~ and used to treat
cancer. The second type of cc,-~ -ds are novel compounds described here for the first time.
30 These novel compounds are also useful when combined with taxol type co~ ounds and used for
the tre3~ment of cancer.
I. Known ComPounds
I. The known ~~ Joullds, and the source of those colllpoullds, are listed below, and
described by name and by l~ ce to the labeled structures.
A) Indolocarbazole Type Compounds
1) The "Fi~t Known Derivatives of K-252." The "First Known Derivatives
~ W O 94/04541 2 1 ~ 0 6 5 ~ - P~-r/US93/07054 ~
of K-252" are all of the compounds disclosed in U.S. patent 4, 877,776. U.S. patent 4, 877,776
incorporated herein by reference.
2) The "Second Known Derivatives of K-252." The "Second Known
Derivatives of K-252" are all of the compounds fii~rlosed in U.S. patent 4,923,986. U.S. p~ent
5 4,923,986 incorporated herein by reference.
3) The specific ~I~pou~ds below are more preferred,
3(a) KT5823
IC~3
[~
C~o o~
3(b) K-252A
0
- 20
H0 H
O=C--O
~3C
3(c) KT5926
~3C~ O~_N/H
~0 ~ C
O=C
C~3
~ WO 94/04541 2 1 4 0 fi 5 3 PCI/US93/07054
3(d) KT5720
0~_N/~
H0 H3C
O=C
CH2
(CH2)3
~CH2
C~2
3(e) Staurûsporine
B. Non-Indolecarbazole Type Co."~,."~
1) Adl iallly~;illS
2) Amilorides
3) Calpl1o~
4) Chlo~L ~u~ 7~
5) The compound known as "HA-1004"
6) IndomethacinS
7) Okadaic acid
8) PhenazocineS
9) Polymyxin Bs
10) 2-~lillopulilleS
I 1) 6-dimethyl-ami-~opu~ineS
12) Sphingosines
1 3) T:~moxif~n
14) Compounds related to tamoxifen such as triphenylethylene antiestrogens
15) Trifluope.~illeS
16) Verapamils
17) 3-isobutyl-1-methyl-x~nthinP.S
1 8) 8-CI-cAMP
Cûl--pou-~ds marked with a superscript s are available from Sigma Ch~ Company.
Taxol and taxotere can be obtained from The National Cancer Instihl~
::
2 1 4 0 6 ~ 3 ~ ~ PCr/US93/070~4 ~
The clinical ~h~rm~cology of taxol is reviewed by ~ric K Rowinsky and Ross C.
Donehower, The Clinical Pharmacology and Use of Antimicrotubule Agents in CancerChemother~eutics, Pha~nac. Ther., Vol 52, pp 35-84, 1991. Clinical and p,ecli";~l studies
with taxol are reviewed by William J. Slirhtonmyer and Daniel D. Von Hoff, Taxol: A New and
5 Effective Anti-cancer Drug, Anti-Cancer Drugs, Vol. 2, pp 519-530, 1991.
Taxol and analogs thereof are the subject of various patents int~h~(lin~ for example, U.S.
Patent Nos. 4,814,470; 4,857,653; 4,942,184; 4,924,011; 4,924,012; 4,960,790; 5,015,744;
5,157,049; 5,059,699; 5,136,060; 4,876,399 as well as PCT Publication No. WO 92/09589,
Eu~upc~l Patent Application 90305845.1 (Publication No. A2 0 400 971), 89400935.6
10 (P lblir~tion No. Al 0 366 841) and 90402333.0 (P~blic~tion No. 0 414 610 Al), 87401669.4
(Al 0 253 739), and PCT Publication Nos. WO 91/17977, WO 91/17976, WO 91/13066, WO
91/13053.
Rebe~c,....ycin is described in: T. Kaneko and H. Wong, Tetrahedron Letters, Vol. 26,
No. 34, pp 4015-4018 (1985).
The ~J.. po~ s known as K252a, K252b, KT5720, KT5823, KT5926, okadaic acid andu~,uorine, are available from Kamiya Biom~lic~l Company, Thon~n-i Oaks, California.
The compounds known as "H-7," "H-9" and "HA-1004" (B4-B6) are available from
Seik~g;~ America, Inc., S~ r~te~l ul~, Florida
Lav~n-ivstin (B8) is available from Gibco BRL.
The co"lpo~ d, Kampferol-7-ne~ s~,idositlP-, is available f~m Apin Ch~mic~l Co.,Abingden, OArol~ ;, United Kingdom.
All of the docllm~nt~ referred to ahove are ineo,l.o,~ed by reference herein.
II. New Compounds
II. The new co~pou,lds of this invention are identified in two ways: by the descriptive
name and by reference to structures cont~in~d in appropriate charts. In some situations, the
proper stereoch~mi~ry is also c~,.sc ~ in the charts.
In this document the parenthetical term (Cn-Cm) is inclusive such that a compound of
(Cl-C8) would include compounds of one to 8 carbons and their isomeric forms. The vanous
carbon moieties are defuned as follows: Alkyl refers to an ~lirh~tic hydrocarbon radical and
includes b,~ ed or ul,brd,lched forms such as methyl, ethyl, n-propyl, i~plul)yl, n-butyl,
isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, and n-
octyl.
Alkoxy as leult;sell~ed by -O-(CI-C8 alkyl) refers to an alkyl radical which is attached to
the rem~in~lPr of the molecule by oxygen and includes branched or unblanched forms such as
methoxy, ethoxy, n-~ poAy, isou.upoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentoxy,
isopentoxy, n-hexoxy, isohexoxy, n-heptoxy, i~oh~ptoxy, and n-octoxy.
21~06~3
WO 94/04541 PCr/US93/07054
(C3-CIû)cycloalkyl refers to a radical of a saturated cyclic hydrocarbon which includes
alkyl-sub~ ~ cycloalkyl, such as cyclopropyl, 2-methylcyclopropyl, 2,2-dimethylcyclopropyl,
2,3 diethyl-;ycloL,-upyl, 2-butylcyclopropyl, cyclobutyl, 2-methylcyclobutyl, 3-propylcyclobutyl,
cyclopentyl, 2,2-dimethylcyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. Each of these
5 moieties may be svl.~lilulP~ as appropriate.
It will be ~pdlt;n~ to those skilled in the art that compounds of this invention may
contain chiral centers. The scope of this invention includes all en~-~liol,,çric or di~lwt;o..-eric
forms of formula I co~p. ul-ds either in pure form or as ~ lu-~;s of e-n~ntiomer.C or dia-
stereomers. The th~.a~eulic properties of the compo~ln~ls may to a greater or lesser degree
10 depend on the stereorh~mictry of a particular c~ -pu~ A
Both organic and illol~ c acids can be employed to form non-toxic pharmaceutically
acceptable acid addition salts of the co",poullds of this illvwllion. Illustrative acids are sulfuric,
nitric, phos~llo,ic, hydrûchloric, citric, acetic, lactic, tarta~ic, palmoic, me~ Ps..lfonic,
e~ lfon:^ sl~lf~rnic~ sucrinic~ cycloh~ lf~nic~ fumaric, maleic, and benzoic acid.
These salts are readily prepared by m~tho-lc known in the art
The co",pou"ds of this invention can be made in acc("d~ce with the processes
described in the PREPARATIONS AND EXAMPLES for the pl~alalion of novel compoundsand illustrated in the GENERAL REACI IONS and the REACTIONS OF CHART A and
CHART B.
In clinical practice the col~ lds of the present invention will normally be
a lminictered by injection, in the form of ph~rm~r~lltic~l p,~ions co",~.isi"g the active
ingredient either as a free base or as a ph~rm:lcPlltic~lly acceptable non-toxic, acid addition salt~
such as the hydrochloride, lactate, acetate, mesylate, meth~neslllfonate, or slllf~m~1~ salt, in
association with a ph~nn~lltically acceptable carrier. The use and ~t1minictration to a patient
to be treated in the clinic would be readily ~pdle.~L to a physician or ph~rm~cict of oldillaly
skill in the a~
In ll-era~el Lical treatment the suitable daily doses of the co.l.poullds of the invention
should fall within the following ranges: Taxol, taxotere and related compounds should be
a~lmini~ered from .001 mg/kg to 10 mg/kg, plcfclably between .05 mg/kg to 5 mg/kg for
intravenous a(1minictration. The compounds to be combin~d with taxol should be ~mini~t~red
in the sarne dosage range. The precise dosage will be aL,~.-l to an ordinarily skilled physician
or pharrnacologist taking into account factors such as the age, weight, sex, and medical
condition of the patient being treated. Also relevant is the potency of the particular compound
and its ability to potentiate the effects of taxol. The potency of the co-"poullds are indicated by
the standard tests described below.
The New Compounds:
WO 94/04541 2 1 ~ 0 6 5 3 PClr/US93/07054
-12-
A colL.poulld of FORMULA I, below,
~ R2
~8 ~
~~ FORMULA I
- R7 H' ~;;,`CH3
R6 (~
- wherein,
Rl is -H, -(Cl-C4 alkyl), -C(O)-(Cl-C4 alkyl), -NH2,
-C(O)-NH2. -CH2CH2-N(Rl-1)2'
wherein Rl l is -H or -(Cl-C4 alkyl),
R2 is -H, or R2 and R3 taken together are (O),
R3 is -H, -OH or R2 and R3 taken together are (O),
R4 is -H, -OH, -NH2, or-O-(Cl-C4 alkyl),
R5 is -OH, -O-(C1-C4 alkyl), or-O-C(O)-(Cl-C4 alkyl),
R6 is -(C6-C12 alkyl), -(C3-C10 cycloalkyl), -(CH2)nCH2N(R6 1)2,
wherein R6 1 is -H, or -(Cl-C4 alkyl),
R7 is -H, or-NH2,
R8 is -Cl, -Br, -H, -CH3, -CH2OH, -OH, -O-(Cl-C4 alkyl),
-N(R8 1)2. or-NHc(o)-NH(R8-l)~
wherein R8 1 is -H or-(Cl-C4 alkyl)
wherein n is 0-5
with the proviso that:
a) when R2 or R3 is -OH then Rl is H,
b) when Rl, R2, R3, R4, and R7 all equal H and R5 is OH
then R6 does NOT equal -(CH2)5CH3,
c) when Rl, R4, and R7 all equal H, and R2 cu",bil,ed with R3 is (O),
- and R5 is OH, then R6 does NOT equal -(CH2)5CH3.
d) when R4 is -OH, -NH2, or -O-(Cl-C4 alkyl), then R4 and R8 are the same.
Preferred ComPounds
The preferred cu-llpoul-ds of this invention are those, r~eferring to the cc,lllpoulld of
FORMULA I, wherein Rl is H or CH3; R2, R3, and R7 is H, R5 is OH or OCH3;
~ WO 94/04541 21 4 0 6 ~ 3 PCI'/US93/070~4
-13-
R8 is -O-(Cl-C4 alkyl). The following coLu~u--ds are preferred, Example B4 and Example
A-l, Example A-l whose structure is shown below.
S CII3~0~ ~
~`CH (Example A-l)
3 ~ 0_~0
Biolo~ical ActivitY
Since taxol is known to be an effective rh~moth~l~ulic agent, for ~Y~mple in theLlcd~ cllL of ovarian cancer, any co-treatment that increases the toxicity of taxol on cancer cells,
such as CHO cells, would be likely to increase the chemull-c ~uLic effect of taxol in cancer
patients or to allow smaller doses of taxol to be ~rlmini~rcd. The coLu~u -ds of this invention
sy~ e with taxol to produce tumor cell toxicity at lower doses than taxol alone, this requires
the conrlllcion that the c~ will be effective in ~yllc~iLillg with taxol in killing tumor
cells in human cancer patients. Additional studies that evaluate the c~ lds effectc on
human breact call MX-l tumors, desrri~ed below also support this conrlncion.
The compounds of this invention are ~l~clcrule useful for the same cancers for which
taxol h~s been shown active, including human ovarian tumors, m~mm~ry tumors, and malignant
m~l~rlnm~ lung tumors, gætric tumors, colon tumors, head and neck tumor., and le--k~mi~
See, e.g., the clinical ph~rrn~ology of taxol is reviewed by Eric K Rowinsky and Ross C.
Duneho~er, The Clinical Pharmacology and Use of Antimicrotubule Agents in CancerChemothel~ulics, Pharmac. Ther., Vol 52, pp 35-84, 1991. Clinical and pre~linir:~l studies
with taxol are reviewed by William J. Slichenmyer and Daniel D. Von Hoff, Taxol: A New and
Effective Anti-cancer Drug, Anti-Cancer Drugs, Vol. 2, pp 519-530, 1991.
Cell lines and ~rowth.
The parental CHO line, lOOOla, is a subclone of the CHO line Pro~5 (Stanley et al.,
1975). The line was m~int~ined in alpha-MEM Earle's Salts supplemented with 2 mMglut:lmin~, 100 units/ml per~icillin, 100 ,ug/ml ~ll~lllycin and 10% fetal bovine serum.
All cell lines were maintained at 37C in 5~o C02 in a h-lmi-lifie~ incubator. Periodically, the
cell lines were tested for mycoplasma and always found to be free of infection.
Compounds were dissolved in dimethylsulfoxide (DMSO) and then diluted into medium for cell
WO 94/04541 2 1 ~ 0 6 ~ 3 PCr/US93/07054 ~
growth assays.
Dru~ sYner~Y e~e,UIlcll~ - "lOOOla" cell lines.
Cells were treated ~im~ ;. ,Pously with the exp~rimental colupo~ d and taxol in 132
different combin-qtions of doses in 96 well plates. l~le 96-well plates were inrubqtpd for four
5 days. Cell growth was det~rminPd by the develvpment of the colorimetric dye 3-(4,5-
Dimethylthiazol-2-yl)-2,5-di~hellylletrazolium br~mide (~I~ as des~ribed by ~osmqnn, 1983.
MTT dissolved in PBS at 2 mg/ml was added to the plates akeady c~ ;.;"i,-g growth medium
to give a final c.,nc~.ll.dlion of 0.2 mg/ml in each well. Plates were then i~ Jbal~d for 3 hours.
The medium co.,~ .i..g MTr i drug was then aspirated off and 100 ,ul/well isop-~l,~.ol
10 ari~iifiçd with 0.04 N HCI was added. Plates were shaken for S minutes and absoll~lce was
read at 570 nm on a Bio-tek EL 312e Bio-kinetics microplate reader.
Percent growth for lOOOla ceUs was ~Ptermin~d by dividing the absolbd-lce reading at
each drug dilution by the reading in control wells. LD50s for each colu~ou,ld were dPtPrminPd
to be the conrentration of drug at which a 509'0 inhibition in cell growth was obtained.
15 potPnhqting effects from the combination of col-lpou,lds on lOOOla cells was detPrminPd by
gl.~ lg the combin~tion~ of drugs which gave LD50s in the form of an isobologram ~llm~n,
1987 and Brunden, 1988).
The effectiveness of combin~tion~ of colll~u..ds with taxol on the killing of wild type,
lOOOla, ceUs is shown by means of an isobologram. The comrolm-l~ of this invention act in a
potentiating or synergistic manner with taxol to kiU cells with much lower doses in comhin~tion
than would be eYrectPd if the drugs were merely eYhibiting additive effects. This effect is
sl~pri~ing and ~ l The isobolograms are displayed as FIGURES 1-21. FIGURES 1-21
de.llol~l~e the effectiveness of combinations of colll~ullds with taxol on the killing of wild
type cells. FIGURE 4 is in~luded in the series to show how a cOI~ ld with no potPnti:lting
effect behaves.
The isobologram shows the effectiveness of a comhin~tion of 2 drugs for the killing of
wild type, lOOOla CHO, cells. The data line is the solid line with open circle or triangle data
points. The data line shows the combin~tion of doses which gives an LD50 for the cells. In
FIGURES 1-21 the concentration of taxol is plotted against the con~Pntration of drug. The
diagonal dashed line shows the predicted ~ncellL ~iolls of drugs if their comhin~tion only had
an additive effect. If any data points were above the dashed line the date would indicate the
combination of compounds had antagonistic effccts. Data points below the line indicate the
co.llpoullds have potenti~ting or synergistic effects. Compare the isobologram in FIGURE 4,
showing NO pot~onti~ting effcct, to the other isobolograms.
In addition to the data provided in the isobolograms the cc,lllpoullds have been tested in
mice. Compound KT5720 has been tested on tumored mice and colu~ul.ds KT5926 and
2140653
~ WO94/04541 ' PCI/US93/07054
-15-
KT5720 have been tested in non-tumored mice.
Dru~ syner~y e~"e-;...c..l~ - MX-I tumors.
FIGURE 22 shows the effect of KT5720 both separately and in com'~ in~tion with taxol
in tumored mice. Human breast cell MX-l tumors were impl~nt~ subcutaneously as 2 mm
5 cubes in athy_ic mice. Mice were dosed every day for five days with drugs or vehicle control.
The vehicle used was 2% dimethyl~t:~mi~e, 10% emul~hor, 88% saline. Animals received
12.5 mg/kg taxol (shown in figure as solid circle data points), 25 mg/kg KT5720 (shown in
figure as open triangle data points), 12.5 mg/kg taxol + 25 mg/kg KT5720 (shown in figure as
solid triangle data points), or vehicle alone (shown in figure as open square data points). Tumor
10 burden was mea~u,~,d every two or three days starting with day 5 and volume was c~ tç~
In 'FIG'LlRE 22 the size of the tumor in millimeters is plotted against time in days. Eight mice
were used per dose group. Results are graphed with standard errors. The results show that there
was no effect of KT5720 alone on inhibition of growth of the tumor ce'lls. Taxol, at 12.5
mg/kg, has a modest effect on reducing the tumor burden in these mice. The combin,.tion of
15 KT5720 plus taxol clearly show a potentiation of the taxol effect by the addition of KT5720. In
summary, KT5720 has no effect by itself, but in comhin^~ion with taxol, at the dosage tested, it
causes a dramatic inhibition of tumor growth.
Dru~ sYner~y e ~ nlC - non-tumored mice.
FIGURE 23 shows the effects of co,lll,ou"ds KT5926 and KT5720 on non-tumored
20 mice. When the ~oul~ul,ds are combined with taxol and then ~ d to non-tumored
normal mice they show a dramatic amount of toxicity. There was no lethality at the doses
shown when these drugs were given individually. This means that there are strong synergistic
effects with the co..lpoullds in vivo. The com'c in~fion of drugs will be effective in tumor
bearing mice and as a cancer treatment for hnm~ c See f~[GURE 23 - IN VIVO EPFECTS.
25 This figure provides a table of data showing the toxicity of several of the drugs combined with
taxol as compared to the individual ~lminictration of the drugs on non-tumored mice.
The required ~.y"e,~i~.lically effective amounts (co~ n~ ions) will vary dep~n-lin~ on
the particular types of individual to be treated ta~cing into consideration various conditions
including age, weight, type of cancer treated, stage of disease, etc. Effective amounts can be
30 readily det~rmin~ by routine expç~.-iment~tiQn
Without fur~her elaboration, it is believed that one skilled in the art can, using the
preceding description, practice the present invention to its fullest extent. The following detailed
examples describe how to prepare the various co"~pou ~ds and/or perform the various processes
of the invention and are to be con~L,ued as merely illustrative, and not limi1~tionc of the
35 preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize
appropriate variations from the procedures both as to re~;l~ll~ and as to reaction conditionc and
~ W O 94/04541 2 1 4 0 6 5 3 PC~r/US93/07054
-16-
tt?chni~ c
PREPARATIONS AND EXAMPLES for the preparation of novel compounds.
GENERAL REACTIONS
S IRl ~
O,~,~N~lR2
R8 ~ ~ ~R4
R7 ~1' ~ CH3
CH30~ R5
Starting m~t~riqlc
Step I
IRl
R8 ~, ~R4
R7 ~ CH3
R6 0~1
Starting ~t.~rj^'~ The starting materials are obtained by using the procedures
described in U.S. Patent 4,923,986 and U.S. Patent 4,877,776. U.S. patents 4,923,986 and
4,877,776 are inco",o.ated by reference into this ~oc~-m~nt
In addition to the patent references above, the starting materials for the reactions are also
30 described in non-patent litel~tult;. The col~)oul.d known as K252A is described in Kase, H., K.
Iwahashi, and Y. Matsuda, K252a, "A Potent Illhi'~ilor of Protein Kinase C *om Microbial
Origin." J. Antiob. (Tolyo) 39:10066-1071 (1986). The compound known as KT 5926, and
related compounds are described in S. N~ nichi~ K. Yamada, K. Iwahasha, K. Kuroda and H.
Kase, "KT5926, a Potent and Selective Inhibitor of Myosin Light Chain Kinase." Molecular
35 Pharmocology, 37-.482488 (1990). The other co-l-poullds of formula 1 where R6 is Cl-Cs alkyl
are described in U.S. Patent 4,923,986 and U.S. patent 4,877,776. ln general, treatment of
21~06~3
WO 94/04541 PCr/US93/070S4
-17-
compounds of formula 1, where R6 is CH3, are treated with R6-OH where (R6 is C6-C12 alkyl)
and KCN to give the desired compounds. All the starting materials are described in the above
patents. Compounds of the type R5 is H are described in WO 91/09034 publi~hed 27 June
1991. All the above dou~..e~ are incorporated by reference herein.
The General Procedure for producing variations for the R6 group is as follows:
To an appropriate starLing material such as KT252a add an alcohol such as n-hexanol
(R6 is -(CH2)5CH3). Stir the mixture at tempe~tul~s ranging from room temperature to 125
degrees until dissolution is complete. An equal weight amount of solid KCN is added and the
reaction mixture is stirred for an additional 18 to 144 hours at tempe,alu,~s ranging from room
telllpe.~ult; to 125 degrees. The reaction mixture is poured into ethyl acetate and the ethyl
acetate solution is extracted with water. The organic solution is dried over anhyd uus sodium
sulfate, filtered and evdpo-aled to dryness under high vacuum at tempe~ ..t;s ranging from 35
to 70C to near dry-ness. Hexane is added to the residue and the resulting solids are allowed to
sit, under hexane, for 24 hours. The solids are filtered and washed well with hexane and dried
15 at 40C to give the desired co~ )ouuld. The materials may be identified by their retention time
on HPLC. Detailed HPLC conditiûns are provided in the examples below. For el~ F'e, when
the following HPLC co~iitic n~ are used the rt;te--lion time for KT252a is 2.54 minut.os HP~C:
Altex Ul~ ,he,t;-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 wal~, ~etol-il.ile; 2 ml/minute).
Wo 94/04541 2 1 4 0 6 ~ 3 PCI /US93/07054
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RE~ACTIONS OF CHART A
~
~CH3 (KT 5926-A)
CH30
Step I
H
C~3~~
\~"` CH
CH3 ~ ~
(Example A- I )
Procedure A. E2~rtion~ of step 1, above. Pl~p~alioll of Example A-l, (R8 is -O-
(CH2)2CH3, R6 is -(CH2)5CH3 ), from the starting material KT-5926.
F.Y~mp'~ A-l is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-
30 10-carhoxylic acid, 2,3,9,10,11,12-hexahydro-l~hydroxy-g- methyl-1-oxo-16-propoxy-, hexyl
ester, (9R-(9.alpha,10.beta, 12.alpha.)).
To 0.8 mg of KT-5926 (0.0015 mmol) is added 0.5 ml of n-hexanol and, after
dissolution is complete, 1.0 mg of KCN is added. The reaction mixture is stirred at room
temperature for 24 hours at which time HPLC inr~ tçs all the star~ng material has reacted.
35 Add 0.5 ml of a 35:65 water :l~etonitrile solution, followed by i~r.eto~ .ile (to complete
dissolution) and chromatograph the entire reaction solution on a p,~dl~ry HPLC system (2-
~ WO 94/04541 2 1 4 0 6 S 3 PCI/US93/07054
-19-
PrepPak 25 X 100 mm Car~idge) microBon~ k C18, 10 microns; 35:65 water acetonitrile at 8
mlhnin taking 24 ml fractions. The fractions co~ g F.x~ A-l, are combined and
e~apo,~ed to dryness to give 0.12 mg of product. HPLC data was run as follows: HPLC: Altex
Ultrasphere-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 ~aLe, ~toni~rile; 2 ml/minute) rt is
5 18.69 minutes for F~ mple A-l; rt is 3.63 minutes for KT 5926.
W0 94/04541 2 1 ~0 ~ 5 3 ` PCl/US93/07054
REACrIONS OF CHART B
S ~
~ " ~3C
O=C--O
~3C (K252a)
Step 1
..
=
Q~N~
~> (Example B-1)
~o `C~3
o=O
C, ~2
( ,CH2)2
,CH2
c~3
Procedure B. Reactions of step 1, above. Preparation of FY~mp'~ B-l, (R8 is H, R6 is
-(CH2)6CH3 ), from the starting m~tPn~l, K252a
Example B-1 is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1 '-kl)pyrrolo(3,~i)(1,6)benzodiazocine-
10~arboxylic acid, 2,3,9,10,11,12-hexahydro- l~hydlu~cy-9-
methyl-l-oxo-, heptyl ester, (9R-(9.alpha,10.beta.,1~ rh~ )).
To 15 mg of KT252a (0.032 mmol) add 2 ml of n-heptanol (R6 is
WO 94/04541 2 1 4 0 6 ~ 3 Pcr/US93~070s4
-21-
-(CH2)6CH3). The mixture is allowed to stir at room temperature until dissolution is complete.
15 mg of KCN is added and the reaction mixture is stirred for an additional 96 hours. The
reaction mixture is poured into 20 ml of ethyl acetate and the ethyl acetate solution is extracted
with water. The organic solution is dried over anhydrous sodium sulfate, filtered and evapor~ted
5 to dryness under high vacuum at 59C to near dryness. 15 ml of hexane is added to the residue
and the resulting solids are allowed to sit, under hexane, for 24 hours. The solids are filtered
and washed well with hexane and dried at 40C to give 7.8 mg of Example B-l. HPLC: Altex
Ul~ hel~-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 wateraretc)nitrile; 2 ml/minute) ~e~ io
time is 7.14 minutes for Example B-l; 2.54 minutes for K252a Mass Spec. theory predicts
10 552.2498 mass units; Measured: 552.2484.
Example B-2, (R6 is -(CH2)7CH3), is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1 '-kl)pyrrolo(3,~i)(1,6)benzodiazocine-
10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hyd,u~y-9-
15 methyl-l-oxo-, octyl ester, (9R-(9.alpha,10.beta.,17 ~Irh~q )).
Using procedure B only sul.~liluli-lg n-octanol (R6 is -(CH2)7CH3) in the reaction
described above, and stirring at room te~perdlu,t; for 120 hours, gives Example B-2, æ an
amber solid. HPLC: Altex Ull.,~ -G-ODS 05 micron, 4.6 mm X 25 cm, (35:65
v~a~..aCelu~iLIile; 2 ml/minute) retention time is 3.325 mimltes Mass Spec. theory predicts
20 566.2655 mæs units; Measured: 566.2652.
FY~mple B-3, (R6 is -(CH2)8CH3), is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4- i)(1,6)benzodiazocine-
10-carboxylic acid. 2,3,9,10,11,12-hexahydro-10-hydroxy-9-
25 methyl-l~xo-, nonyl ester, (9R-(9.alpha.,10.beta,1~ ~Iph~ )).
Using procedure B ûnly s~ ilu~ g n-nonanol (R6 is -(CH2)8CH3) in the reaction
described abûve, and s~rring at roûm te~ e~ul~ fûr 144 hûurs, gives Fx~mrle B-3, as an
amber solid. HPLC: Altex Ul~ here-ODS 0.5 micron, 4.6 mm X 25 cm, (25:75
water ~cet-~nitrile; 2 ml/minute) retention time is 6.54 mimltPs Mass Spec. theory predicts
30 580.2811 mass units; Measured: 580.2819
.
Example B-4, (R6 is -CH(CH2CH3)((CH2)3CH3). is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1 '-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-
10-c~boxylic acid, 2,3,9,10,11,12-hexahydro- 10-hydroxy-9-
35 methyl-l-oxo-, I-ethylpentyl ester.
Using procedure B only ~ul,~ "i..g 3-heptanol (R6 is -CH(CH2CH3)((CH2)3CH3) in
W O 94/04541 2 ~ 3 PC~r/US93/07054
-22-
the reaction described above, stirring at 110 degrees for 144 hours, gives Example B-4, as an
amber solid. HPLC: Altex Ull,~he-t-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65
water aretonitrile; 2 mlhninute) retention time is 6.44 minntP~c Mass Spec. theory predicts
552.2498 mass units; Measured: 552.2501.
FY~mple B-5, (R6 is -CHCH3(CH2~4~H3), is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1 '-kl)pyrrolo(3,4- i)(l,6)ben70~ 70rin.o-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-l~llydlu~y-9-
methyl-l-oxo-, 2-methylhexyl ester.
Using ploce(lul~; B only s~ g 2-heptanol (R6 is -CHCH3(CH2)4CH3) in the
reaction dt~scribed above, and heating at 100 degrees for 18 hours, gives FY'-1~ ' B-S, as an
amber solid. HPLC r~,~,.liull time is 6.67 minllt- c Mass Spec. theory predicts 552.2498 mass
units; Measured: 552.2501.
. ;e B-6, (R6 is -CHCH3(CH2)5CH3), is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4- i)(l~6)ben7or1;~
10-carboxylic acid, 2,3,9,10,11,12-hexahydr~10-l~y-l,u~y-9-
methyl-l-oxo-, 2-mc;Ll.yllle~sLyl ester.
Using procedure B only s~ g 2~ctanol (R6 is -CHCH3(CH2)5CH3) in the
20 reaction described above, stirring at 100 degrees for 96 hours, gives FY~m~'o B-6, as an amber
solid. HPLC: Altex Ultrasphere-ODS Q5 micron, 4.6 mm X 25 cm, (25:75 ~dlc..acetoni~.ile; 2
mlhninute) retention time is 4.55 minllt~c. Mass Spec. theory predicts 566.2655 mass units;
Measured: 566.2652.
ple B-7, (R6 is -CHCH3(CH2)6CH3), is named:
9,12-Epoxy-lH-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)bPn7Or~i~7nçine
10-carboxylic acid, 2,3,9,10,11,12-hexahydro-l~hydroxy-9-
methyl-l-oxo-, 2-methylocql ester, (9R-(9.alpha,10.beta,12.alpha)).
Using procedure B only ~ul,~;l..li.~g 2-nonanol (~6 is -CHCH3(CH2)6CH3) in the
30 reaction described above, stirring at 100 degrees for 96 hours, gives F.Y~Tnp'~ B-7, as an amber
solid. HPLC: Altex UlL~L,hert;-ODS 0.5 micron, 4.6 mm X 25 cm, (25:75 water acetonitrile; 2
ml/minute) retention time is 6.12 minut~c Mass Spec. theory predicts 580.2811 mass units;
Measured: 580.2797.
3s FY~mrle B-8, (R6 is -(CH2)20CH2CH3), is named:
9,12-Epoxy- l H-diindolo(1,2,3-fg:3 '~2 ',1 '-kl)pyrrolo(3,4- i)( l ~6)be n
WO 94/04S41 2 1 4 0 6 5 3 PCr/US93/07054
-23-
10-call o~sylic acid, 2,3,9,10,11,12-hexahydro- 10-hyd~y-9-
methyl- I -oxo-,2-ethoxyethyl ester, (9R-(9.alpha.,10.beta..12.alpha.)).
Substihlting ethoxyethanol (R6 is -(CH2)20CH2CH3) in the reaction described above,
and stirring at room temperature for 96 hours, followed by 50 degrees for 24 hours, gives
S Example B-8, as an amber solid. HPLG: Altex Ul~ el~;-ODS 0.5 micron, 4.6 mm X 25 cm,
(35:65 water:~netoni~rile; 2 ml/minute) retention time is 1.54 minutes Mass Spec. theory
predicts 526.1978 mass units; Mea~u,t;d: 526.1959.
.
Example B-9, (R6 is -CH(cyclo-CH2)5), is named:
9~l2-Epoxy-lH-diindolo(l~2~3-fg:3~2~ )pyrr~lo(3~4-i)(l~6)b~n
10-c.~ ylic acid, 2,3,9,10,11,12-hexahydro-10-l~yd,uxy-9-
methyl-l-oxo-, cyclohexyl ester, (9R-(9.alpha,10.beta.,1~ ~Iph~ )).
Sul.~ il-g cyclohexanol (R6 is -CH(cyclo-CH2)5) in the reaction described above,stirring at 80 degrees for 18 hours, gives FY~mF" B-9, as an amber solid. HPLC: Altex
15 Ultr~cph~re-ODS 0.5 micron, 4.6 mm X 25 cm, (35:65 water-~cetonitrile; 2 mlhl~inute) retention
time is 3.95 minllt~s Mass Spec. theory predicts 536.2185 mass units; Me~ul~d: 536.2169.
WO 94/04541 2 1 ~0 6 5 ~ 't '~ PCI/US93/07054
-24-
TABL~ 1 - IN VIVO E~FFECTS
mg/kg Dose Day of Dea~h
Mouse #1 Mouse #2
Taxol 10.0 ~-- --
5 .0
KT5926 15.0 -- --
5.0
1.0 -- --
0.1 -- --
Taxol + KT5926 10.0 + 15.0 10 11
10.0+ 5.0 7 10
10.0 + 1.0 9 9
10.0+0.1 9 10
5.0+ 15.0 9 14
5.0+ 5.0 9 10
5.0 + 1.0 8 9
5.0+0.1 10 11
BDFI mice
Two mice per dose (mouse #1 and mouse #2)
20 Drugs were given i~ il(Jne~lly on days 1-9
* No day of death given means that the mice did not die dunng the duration of the
experiment.
